Fluorescence Lifetime

Fluorescence excited-state lifetime imaging

Time-resolved
fluorescence spectroscopy is a well-established technique for studying
the emission dynamics of fluorescent molecules i.e. the distribution of
times between the electronic excitation of a fluorophore and the
radiative decay of the electron from the excited stated producing an
emitted photon. The temporal extent of this distribution is referred to
as the fluorescence lifetime of the molecule. Lifetime measurements can
yield information about the molecular microenvironment of a fluorescent
molecule. Factors such as ionic strength, hydrophobicity, oxygen
concentration, binding to macromolecules, and the proximity of molecules
that can deplete the excited state by resonance energy transfer can all
modify the lifetime of a fluorophore. Measurements of lifetimes can
therefore be used as indicators of these parameters. Furthermore, these
measurements are generally absolute, being independent of the
concentration of the fluorophore. This can have considerable practical
advantages. For example, the intracellular concentrations of a variety
of ions can be measured in vivo by fluorescence lifetime
techniques (Szmacinski et al., 1994 Methods Enzymol. 240, 723). Many
popular, visible wavelength calcium indicators, such as Calcium Green 1,
give changes of fluorescence intensity upon binding calcium. The
intensity-based calibration of these indicators is difficult and prone
to errors. However, many dyes exhibit useful lifetime changes on calcium
binding and therefore can be used with lifetime measurements (Lakowicz,
et al., 1994 Cell Calcium 15, 7). This gives the considerable advantage
that absolute measurements of concentration can be made with no
elaborate calibration procedures required. Alternatively, lifetime
measurements may be used to calibrate the intensity signals from these
indicators when maximum sensitivity is required.

An exciting new
development of the field has been the development of the technique of
fluorescence lifetime imaging microscopy (Lakowicz et al., 1992 Anal.
Biochem 202: 316; Wang et al., 1992. Crit. Rev. Anal. Chem. 23: 369;
Gadella et al., 1993. J. Cell Biol. 129, 1543). In this technique
lifetimes are measured at each pixel and displayed as contrast. Lifetime
imaging systems have been demonstrated using wide-field (Lakowicz et
al., 1992 Anal. Biochem 202: 316), confocal (Sanders et al., 1995 Anal.
Biochem. 227: 302), and multiphoton (French et al., 1997. J. Microsc.
185: 339) imaging modes. FLIM combines the advantages of lifetime
spectroscopy with fluorescence microscopy by revealing the spatial
distribution of a fluorescent molecule together with information about
its microenvironment. This extra dimension of information can be used to
discriminate among multiple labels on the basis of lifetime as well as
spectra. This would allow more labels to be discriminated simultaneously
than by spectra alone in applications where many labels are required
such as FISH. There are also promising applications of lifetime imaging
in the medical sciences. For example, tumors have been detected in mice
sensitized with a hematoporphyrin derivative by lifetime imaging
(Cubeddu et al., 1997 Photochem Photobiol 66(2):229).

We are
particularly interested in the possibilities that are offered by
multiphoton lifetime imaging of live specimens. In these applications
lifetime imaging, in conjunction with spectral imaging, should greatly
facilitate studies using ion indicator probes and FRET studies of
intermolecular distances. For example, a remarkable calcium indicator
has recently been described that is a chimeric protein based on two
spectrally distinct forms of fluorescent protein (cyan and yellow) and a
calmodulin molecule (Miyawaki et al., 1997 Nature 388: 882). Being a
naturally fluorescent protein, genetic transformants can be made so that
transformed animals will express the indicator in a range of cell types
determined by the promoter. The excitation wavelength is chosen to
excite the cyan fluorophore. On binding calcium, the calmodulin portion
of the molecule changes conformation bringing the two fluorophore
regions closer together allowing resonant energy transfer between the
cyan and the yellow. This will cause a shift of the emitted spectrum
from cyan to yellow. The development of this engineered protein (known
as Cameleon) is a remarkable development as it circumvents all the
problems associated with loading probes into cells since stable
transgenic lines expressing Cameleon can be used. However, one of the
problems with Cameleon is although ratiometric methods can be used, the
signal change on binding calcium is quite small making this indicator
less sensitive than other indicators such as Calcium Green. Lifetime
measurements are a sensitive indicator of FRET (Godella et al., 1995. J.
Cell Biol. 129, 1543) and in combination with spectral measurements
should provide a more sensitive indication of calcium levels.

Techniques for lifetime imaging

Fluorescent
lifetimes can be measured either in the frequency domain or in the
temporal domain. Three general strategies have been used to measure
fluorescence lifetimes:

Frequency-domain imaging

In this
scheme a high-frequency, modulated light source is used for fluorophore
excitation. By the use of a gain-modulated detector, the phase shift and
amplitude demodulation of the fluorescence signal is determined. From
these data the fluorescent lifetime of the probe can be calculated. This
scheme is robust and has been extensively used (Wang et al., 1992).
However for our purposes it suffers from several drawbacks: the detector
is only working at 50% of its maximum efficiency because it is gain
modulated, several data sets taken at different excitation modulation
frequencies have to be taken in order to separate two or more lifetime
components, and this scheme does not work well with photon counting
techniques which we favor.

Time-domain lifetime imaging with gated detectors

In
this scheme a gated micro-channel plate image intensifier is used in
conjunction with a CCD imaging camera (e.g. Straub and Hell, 1998.
Applied Physics Letters 73:1769). Spectral information is obtained by
gating the image intensifier on for a narrow time-window at
progressively later intervals after the excitation pulse in a succession
of data frame captures. This scheme is probably the simplest way of
implementing a life-time imaging system. However, it suffers from two
major drawbacks for our application. The method has very poor photon
utilization as only one temporal interval is detected at a time. If
there are 32 intervals for example, 31/32 of the signal is not utilized
and 32 separate frames have to be captured. The second reason this
scheme is not appropriate for a multiphoton imaging application is that
an imaging (i.e. area) detector is used. This means that the deep
sectioning advantage of multiphoton imaging are not fully realized
because scattered fluorescence emission photons will give rise to
background noise rather than contributing to the signal as can be done
with a point-scanning multiphoton system.

Time-domain lifetime imaging with photon counting

For working at low-light levels, photon-counting detectors have
considerable advantages in that they can virtually eliminate noise
contributions from electronic amplifiers or electron multiplier noise in
a photomultiplier. Also, photon-counting systems provide quantized
pulses for every detected photon, allowing the lifetimes to be measured
directly using electronic circuitry. Because of the very high speeds
necessary to obtain sub-nanosecond temporal resolution., time-to-voltage
converters are usually used to measure the interval between the
fluorophore excitation pulse and the time of detection of the emitted
fluorescent photon. Such schemes have been successfully used in
practical photon-counting lifetime detectors (Kelly et al., 1997. Rev.
Sci. Instrum 66(6):2279). These schemes are attractive because of their
efficient utilization of detected photons. However they suffer from
dynamic range problems that arise out of limited counting speeds.
Typically, a time-to-voltage converter together with an associated
analogue (voltage) to digital converter would have a maximum counting
rate of around 1Mhz. Also, with this scheme, only one photon can be
measured in the interval between laser pulses. These limitations
restrict the use of this technique to low light levels when fairly long
exposure times are needed in order to obtain sufficient counts for
accurate representation of the decay curve. The comparatively large
dead-time of this technique can have more insidious consequences.
Immediately after the laser pulse, photons will be emitted at the
highest rate and therefore more will be preferentially lost at this time
because of the dead-time of the detector. This effect can distort the
shape of the decay profile.